The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor (GPCR) that transduces signals from two related signaling molecules that have distinct functions in bone biology: parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) [(1); reviewed in (2, 3)]. PTH is an 84-amino acid polypeptide endocrine hormone that is produced by the parathyroid glands and secreted into the circulation in response to low calcium levels [reviewed in (4-6)]. The classical actions of PTH are mediated by PTH1R expressed in bone and kidney tissues and include stimulation of osteoclastic bone resorption to maintain calcium homeostasis, and stimulation of calcium reabsorption, synthesis of 1,25-dihydroxyvitamin D3, and phosphate excretion in kidney. Paradoxically, PTH also has stimulatory effects on osteoblastic cells resulting in bone formation [reviewed in (7)], providing the molecular basis for the clinical use of PTH as an anabolic therapy for osteoporosis (8). Anabolic PTH therapy requires intermittent administration to avoid the bone resportive effects that predominate with sustained elevation of PTH in the circulation.
PTHrP is a 141-amino acid polypeptide that was originally isolated as the factor responsible for humoral hypercalcemia of malignancy (9-12) and was subsequently shown to be a critical developmental factor that regulates endochondral bone formation [(13, 14); reviewed in (15)]. PTHrP is produced locally and functions in a paracrine/autocrine fashion to activate PTH1R expressed on chondrocytes to regulate their proliferation and differentiation. PTHrP also has anabolic effects when administered to osteoporosis patients (16), but appears to be more purely anabolic than PTH by uncoupling bone formation from bone resorption (17).
PTH and PTHrP can activate several downstream signaling pathways through PTH1R to mediate their effects, but activation of the cAMP/PKA pathway via Gαs-coupling predominates and is responsible for the bone anabolic effect (18). The N-terminal 34-residue peptide fragments of PTH and PTHrP are sufficient to bind and activate PTH1R to the same extent as the native molecules, and PTH- and PTHrP-(1-34) are equally potent for activating cAMP signaling (1). Their interaction with the receptor follows a “two-domain” model. Residues 1-14 interact with the 7-transmembrane (7-TM) helical domain embedded in the membrane and residues 15-34 interact with the N-terminal extracellular domain (ECD) of the receptor (19, 20). The 1-14 fragments of PTH and PTHrP have eight amino acid sequence identities reflecting the critical role this fragment plays in activating the receptor (20). The 15-34 fragments impart high affinity binding to the receptor, but this portion of PTH and PTHrP is less conserved with only three amino acid identities. PTH and PTHrP form similar α-helical structures in solution (21, 22).
Despite their shared two-domain receptor binding mechanism, common secondary structure, and equipotent activation of signaling, PTH and PTHrP differ in their abilities to bind to two pharmacologically distinct PTH1R conformations that are distinguished by the presence or absence of G protein-coupling. The peptides bind with similar high affinity to the G protein-coupled receptor (RG conformational state), but in the absence of G protein coupling (R0 conformational state) PTHrP binding is significantly diminished whereas PTH binding is only slightly decreased (23-25). Thus, PTHrP is more RG-selective than PTH. The different R0/RG selectivity profiles of PTH and PTHrP correlate with distinct temporal effects on cAMP signaling. PTH elicits a longer lasting cAMP signal after ligand wash-out than PTHrP (23). Divergent residue 5 (Ile in PTH, His in PTHrP) is a key determinant of the R0/RG selectivity differences of the peptides (23, 25), but the 15-34 fragment of PTH contributes to its strong R0 binding (23, 26) suggesting that interactions with the ECD contribute to the duration of cAMP signaling. Importantly, temporal differences in cAMP signaling can have dramatic effects in vivo. Compared to wild type PTH, PTH analogs that exhibit increased R0 binding induce sustained cAMP responses in cells and result in increased trabecular bone volume and increased cortical bone resorption in mice receiving daily injections (26). These studies indicate that the R0/RG selectivity profiles of PTH and PTHrP contribute to the different physiological and therapeutic actions of the peptides, and highlight the importance of a detailed understanding of the structural basis of how the binding of PTH and PTHrP to the receptor differs.
The inventors previously developed methodology that allowed them to determine the high resolution crystal structure of the PTH1R ECD in complex with the C-terminally amidated 15-34 fragment of PTH [PTH(15-34)NH2] (27). The ECD was expressed in E. coli as a fusion protein linked to the C-terminus of bacterial maltose binding protein (MBP). MBP solubilizes the ECD during an in vitro disulfide shuffling reaction required to properly form three disulfide bonds and facilitates crystallization of the fusion protein. The PTH1R ECD forms a fold that is conserved among class B GPCR ECDs (28-30) and consists of an N-terminal α-helix followed by two anti-parallel β-sheets and a short C-terminal α-helix, all held together by the three disulfide bonds. PTH(15-34)NH2 forms an amphipathic α-helix that binds to a hydrophobic groove at the interface of the secondary structure elements.